We propose to continue our collaborative study of the red cell and its constituents in normal and diseased state aiming at elucidating the molecular nature of abnormalities such as sickle cell disease. We will continue our investigations of transient and permanent changes in membrane due to changes in ion fluxes caused by injuries to the cell. We will focus on the role of calcium ions and of spectrin using ionophores as experimental models for mimicking cell damage. We will use electronmicroscopy and X-ray microprobes to characterize the state of the membrane and the topography of elements throughout the normal and abnormal cell. In parallel we will carry out viscoelastic measurements of the different cell populations. We will attempt using calorimetry and isotope exchange kinetics to arrive at a thermodynamic description of energy of association between dimers and tetramers in normal and gelling hemoglobin. We are in progress of obtaining extremely precise information about the different steps of ligand binding using isonitriles as ligands to hemoglobin. We also continue our efforts to correlate hemoglobin function with the data obtained by magnetic circular dichroism, sensitive to spin state and the immediate surroundings of iron atom. Finally we will investigate systematically the functional effects and toxicity of the different bifunctional protein reagents when used as antisickling agents.